Identification of 5-HT2A receptor signaling pathways associated with psychedelic potential

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT2A-Gq/11 and β-arrestin2 transducers, making their respective roles unclear. To elucidate this, we develop a series of 5-HT2A-selective ligands with varying Gq efficacies, including β-arrestin-biased ligands. We show that 5-HT2A-Gq but not 5-HT2A-β-arrestin2 recruitment efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT2A Gq-efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT2A agonists. We also demonstrate that β-arrestin-biased 5-HT2A receptor agonists block psychedelic effects and induce receptor downregulation and tachyphylaxis. Overall, 5-HT2A receptor Gq-signaling can be fine-tuned to generate ligands distinct from classical psychedelics.

psychedelic from therapeutic properties 5,6 .Nevertheless, fundamental questions exist regarding which receptors and signaling pathways mediate effects of psychedelics, limiting the rational design of new drugs.
Serotonergic psychedelics are derived from multiple chemical scaffolds (e.g., tryptamines, phenethylamines, and lysergamides), all which activate the 5-HT 2A receptor (5-HT 2A R), a G protein-coupled receptor (GPCR).5-HT 2A Rs appear to primarily mediate psychedelic experiences supported by evidence that the 5-HT 2A R antagonist ketanserin attenuates subjective effects of psilocybin and LSD in humans 7,8 .Psychedelics have also been studied in preclinical behavioral models, including the head-twitch response (HTR), which is a 5-HT 2A R-mediated involuntary head movement in mice that predicts human psychedelic activity 9 .Multiple receptors, however, appear to contribute to the behavioral effects of psychedelics, including psilocybin 10 and LSD 11 , which adds to their complicated psychopharmacology.
Ligands targeting GPCRs stabilize certain receptor conformations that energetically favor coupling to transducer proteins 12,13 .Liganddependent bias has important implications for drug development and clinical pharmacology 14,15 .For example, the G protein-biased μ-opioid receptor (MOR) agonist oliceridine reportedly has a correspondingly improved tolerability profile 16 , although alternative explanations exist 17 .As existing psychedelics activate both Gq and β-arrestin2 via 5-HT 2A R 18,19 , the role these pathways play in the effects of psychedelics is unclear.Although putative non-psychedelic 5-HT 2A R agonists exist 5,6 , an adequate explanation for lack of psychedelic action does not exist.A clear and defined pharmacological signaling mechanism explaining why certain 5-HT 2A R agonists lack psychedelic effects is thus needed.
Herein, we utilized structure-inspired design to develop 5-HT 2A R-selective ligands acting as biased agonists and leveraged these to develop a mechanistic and molecular explanation of biased 5-HT 2A R agonism.Furthermore, these compounds were used to probe the relationship between 5-HT 2A R-Gq versus 5-HT 2A R-β-arrestin activity and psychedelic potential in vivo.Our goal was to identify the 5-HT 2A R transducers mediating psychedelic activity for the rational design of next-generation 5-HT 2A R agonists.

Results
Psychedelics exhibit similar Gq and β-arrestin2 activity at 5-HT 2A R To investigate the 5-HT 2A R signaling profiles of classical psychedelics, we used bioluminescence resonance energy transfer approaches (BRET, Fig. 1A, B, Supplementary Table 1), which provide a proximity measure of intracellular transducer engagement.BRET is used extensively to quantify GPCR-biased agonism and has the advantage of not being susceptible to second messenger amplification or receptor reserve issues obfuscating GPCR signaling preference determinations 20 .BRET has been used to measure 5-HT 2A R-β-arrestin2 and 5-HT 2A R-Gq activity directly 11 , and to confirm 5-HT 2A R G protein coupling preferences 18 .In our assay platform, 5-HT 2A R strongly couples to Gq/11 and β-arrestin2 over all other G protein subtypes and β-arrestin1 (Fig. 1C).
Next, we tested classical psychedelics from multiple chemical classes and examined their effects on Gq dissociation and β-arrestin2 recruitment (Fig. 1D-K).Kinetic issues can confound studies of liganddirected bias 21 , and slow ligand kinetics can delay full receptor occupancy, as observed for LSD at 5-HT 2A R 19 .Therefore, we thoroughly assessed Gq and β-arrestin2 activities at various time points at 37 °C to ensure full receptor occupancy and confirm that transducer preferences do not substantially change when compared at the same time point.Our results show that psychedelics exhibit dynamic, timedependent profiles of Gq and β-arrestin activity that in some cases exceed the activity of 5-HT (i.e., superagonism) at longer time points (e.g., 300 minutes, Supplementary Fig. 1A-H), which is not surprising given the dynamic temporal nature of GPCR signaling 22 .For all tested psychedelics, however, effects on Gq and β-arrestin activity were strikingly similar at equivalent time points and closely mirrored the pathway-balanced endogenous agonist 5-HT (Fig. 1L), indicating no strong preferences for one of the transducers.Psilocin, DMT and 2C-I showed slightly less β-arrestin2 efficacy compared to Gq activity, but the difference was not substantial when both transducers were compared at each respective time point (Supplementary Fig. 1B, C, E).Importantly, all tested psychedelics lacked a strong preference for Gq or β-arrestin2, demonstrating these compounds are not substantially biased for either transducer.

Rational design of a 5-HT 2A -selective agonist template
To engineer a series of biased agonists for interrogating the 5-HT 2A R-coupled signaling pathways associated with psychedelic potential, a scaffold exhibiting some degree of selectivity for 5-HT 2A R over other 5-HT receptors is required.Most psychedelics are not selective for 5-HT 2A R, exhibiting complex polypharmacology 11 .5-HT 2A R shares considerable homology with 5-HT 2B R and 5-HT 2C R, making it challenging to develop selective 5-HT 2A R agonists.To date, few 5-HT 2A R-selective agonists have been discovered, but the N-benzyl-phenethylamines 25CN-NBOH 23 and DMBMPP 24 are purported examples.Importantly, recently discovered "non-psychedelic" 5-HT 2A R agonists show little selectivity for 5-HT 2A R 5,6 , complicating attempts to interpret their psychopharmacology.
To optimize 5-HT 2A R affinity and selectivity, we synthesized a series of 25N-NB analogs designed to modify the electrostatic properties of the N-benzyl ring system (Supplementary Fig. 2, 12; Supplementary Table 2; Supplementary Data 1, 2, and 3).The increased 5-HT 2A R affinity of N-benzyl-phenethylamines is thought to result in part from hydrogen bonding between the N-benzyl 2-position and residues in 5-HT 2A R 28 , but we developed an alternate hypothesis that ring-electrostatics (i.e. the effect of increasing π-electron density in portions of the N-benzyl-ring, quantified as Hammett σ constants relative to C 5' ) drives 5-HT 2A R affinity (Fig. 2B, Supplementary Fig. 3; Supplementary Table 3).Using the 25N series, we found that estimates of increasing electron density around the N-benzyl C 5' position (para to the 2'-position) increase 5-HT 2A R binding affinity and agonist potency (Supplementary Fig. 3; Supplementary Tables 3-8).To confirm the importance of this ring-region, we tested the effect of adding a methoxy group to the C 5' position of 25N-NBOMe (4), which this analog 25N-NB-2,5-DiMeO (20) had reduced affinity 400-fold, suggesting steric clash and/or altered electronics disrupted the optimal electrostatic interaction (Supplementary Fig. 3J).The electrostaticrelationship was supported by correlations with additional potency estimates, experimental NMR chemical shifts, and in silico Hirshfeld surface analyses (Supplementary Fig. 3A-I, K-P, Supplementary Tables 4-6).This resulted in the discovery of several high-affinity 5-HT 2A R agonists within the N-benzyl-phenethylamine class of psychedelics (Supplementary Tables 7, 8, 13).
GPCR crystal and cryo-EM structures only show a "snapshot" of activation and are highly dependent on their ternary complex composition and intracellular binding partners.Therefore, we performed molecular dynamics (MD) to investigate interactions between 25N-N1-Nap (16) and 5-HT 2A R, focusing especially on residue W336 6.48 , and compared the results to simulations performed with 25CN-NBOH in the cryo-EM structure (Supplementary Data 4-7).Simulations of 250 ns were carried out for each ligand, following a common protocol for system preparation and equilibration.In addition, twelve replicate trajectories were computed for each ligand, initiated from the same starting structures, but with independent assignments of initial random atomic velocities, allowing for expanded sampling of configurations and independent validation of our analyses.
To verify the role of W336 6.48 in 5-HT 2A R biased agonism, we constructed conservative mutations, W336 6.48 Y and W336 6.48 L, designed to increase space and accommodate the larger substituents in 25N βarrestin-biased ligands.Although the W336 6.48 L mutant exhibited reduced Gq dissociation and impaired β-arrestin recruitment (Supplementary Fig. 6I) preventing further experimental use, the W336 6.48 Y mutant showed robust Gq and β-arrestin recruitment and was used in subsequent experiments (Supplementary Fig. 6I).When 25N-N1-Nap (16) and 25N-NBPh (17) were tested at the W336 6.48 Y mutant, the Fig. 2 | Rational design of a 5-HT 2A -selective agonist template.A N-Benzylation of 25N (1) to 25N-NB (2) leads to reduced 5-HT 2B receptor efficacy, as measured by Gq dissociation by BRET.Data represent the mean and SEM from three independent experiments performed at 37 °C with 60-minute compound incubation.B Role of N-benzyl ring electrostatics in 5-HT 2A receptor potency leading to development of 25N-NB-2-OH-3-Me (18) using QSAR correlation between 5-HT 2A receptor pK i and Hammett σ constant values (Pearson's R = −0.8887,R 2 = 0.7897, 2-tailed p < 0.0001, N = 15).C The relationship between steric bulk and 5-HT 2A/2C receptor selectivity is shown for the halogen series, leading to the identification of the 5-HT 2A receptor-selective agonist 25N-NBI (10) (left).Also shown is a 5-HT 2A/2C receptor selectivity heatmap comparing the 25N halogen series to 25CN-NBOH (right).D Comparison of 5-HT 2A receptor (green) 5-HT 2B receptor (red) and 5-HT 2C receptor (purple) Gq dissociation activities for 25N-NBI (10) (left) and 25CN-NBOH (right).Data represent mean and SEM from three independent experiments, which were performed at 37 °C with 60-minute compound incubation.E 5-HT 2A receptor Gq dissociation and β-arrestin2 BRET concentration response curves for 25N-NBOH (3, top) and 25N-NB-2-OH-3-Me (18, bottom) showing addition of a 3-methyl group leads to reduced Gq-efficacy.Data represent mean and SEM from three independent experiments, which were performed at 37 °C with 60-minute compound incubation.F 25N-NBI (10) induced fit docking (IFD) with orthosteric site residue side chains displayed.The window shows a zoom-in view illustrating key ligandresidue interactions within the orthosteric site and illustrating the close proximity of the 2'-and 3'-positions to TM6 and TM7, which are known to influence ligand bias.G Summary of structure-activity relationships (SAR) for the 25N series encompassing key effects on electrostatics, 5-HT 2A receptor selectivity, and reduced Gq E MAX .Source data are provided as a Source Data file.
Gq-efficacy recovered substantially, resulting in balanced agonist activity similar to 25N-NBOMe (4) (Fig. 3F).The recovery of Gq activity likely results from the increased space created by a smaller size Tyr residue at position 6.48, allowing the "toggle switch" to move dynamically to initiate Gq-bound activation states.The mutagenesis data support the hypothesis that the W366 6.48 toggle switch plays an important role in the biased agonism of 25N-N1-Nap (16) and 25N-NBPh (17), with steric bulk on the N-benzyl ring forcing W336 6.48 into a unique rotamer conformation, making it less likely that Gq-bound conformations will arise while preserving β-arrestin-preferring conformational states.

β-Arrestin-biased 5-HT 2A agonists lack psychedelic potential
The HTR is commonly used as a behavioral proxy for psychedelic effects because non-psychedelic 5-HT 2A R agonists do not induce head twitches 36 , and because there is a robust correlation between HTR activity in mice and potency to induce psychedelic effects in humans and discriminative stimulus effects in rats 9 .To assess psychedelic potential for the 25N series, seventeen compounds were tested in the HTR assay based on their diverse range of Gq and β-arrestin efficacies.Male mice were used to maintain consistency with the validation experiments supporting use of the HTR as a cross-species readout of psychedelic potential and to leverage the large dataset of HTR doseresponse data previously generated 9,36 .Eleven of the compounds increased HTR counts significantly over baseline levels (Supplementary Tables 15 and 16).Not surprisingly, 25N-NBOMe (4), which acts as a psychedelic in humans 37 , produced a potent HTR response consistent with this effect (Fig. 4A).By contrast, the β-arrestin2-biased compounds 25N-N1-Nap (16), 25N-NBPh (17) and 25N-NB-2-OH-3-Me (18) failed to induce the HTR (Fig. 4B-D; Supplementary Tables 15, 16), suggesting Gq-efficacy is necessary for psychedelic potential.To confirm the β-arrestin2-biased agonists can partition into the brain and engage central 5-HT 2A R necessary for the HTR, we pretreated mice with those compounds, which subsequently blocked the HTR induced by psychedelic 5-HT 2A R agonist DOI (Fig. 4E-H).25N-NBPh (17) had lower potency than 25N-N1-Nap (16) in the blockade experiments, which we believe is due to pharmacokinetic differences limiting its CNS distribution, potentially reflecting its higher cLogP (4.8 vs. 4.5, respectively), thus we focused on 25N-N1-Nap (16)  5-HT 2A R-selective antagonist M100907 (Fig. 4I).Given the potential for species differences, which is evident for rodent versus human 5-HT 2A R 38,39 , we verified that β-arrestin bias exhibited by 25N-N1-Nap (16) and 25N-NBPh ( 17) is preserved at mouse 5-HT 2A R (Supplementary Fig. 9A).
To confirm β-arrestin-biased 5-HT 2A R agonists do not induce the HTR and that this profile is not specific to the 25N series, we synthesized and tested N-naphthyl and N-biphenyl derivatives of other phenethylamine 5-HT 2A R agonists (Supplementary Fig. 13, 14).Similar to previous β-arrestin-biased compounds, these compounds exhibited weak Gq-efficacy but produced robust β-arrestin2 recruitment efficacy (Supplementary Fig. 9B; Supplementary Table 13-16).When evaluated in mice, 25O-N1-Nap (28) and 2C2-N1-Nap (29) failed to induce head twitches (Supplementary Fig. 9C) but fully blocked the HTR induced by DOI (Supplementary Fig. 9D), indicating brain penetration.In summary, five different β-arrestin2-biased 5-HT 2A R agonists did not induce the HTR when tested at doses that block the response to a balanced agonist, confirming this strategy can consistently generate β-arrestin2biased 5-HT 2A R agonists devoid of psychedelic potential.

5-HT 2A -Gq signaling predicts psychedelic potential
Although the 5-HT 2A R signaling pathways associated with psychedelic potential have been previously investigated, the results were inconclusive [40][41][42] .To assess further the involvement of 5-HT 2A R Gq and β-arrestin2 activity in the HTR, correlation analyses were performed using the 25N series, which contains a mixture of HTR-active and inactive compounds.For the 25N compounds with Gq and β-arrestin2 data (n = 14), there was the robust correlation between HTR magnitude (the maximum number of HTR induced by each drug in counts/minute) and 5-HT 2A R Gq-efficacy (%5-HT E MAX ; R S = 0.8242, p = 0.0005; Fig. 5A).By contrast, HTR magnitude was not correlated with β-arrestin2 recruitment (R S = −0.01538,p = 0.9638; Fig. 5B).Notably, the relationship between 5-HT 2A R Gq-efficacy and HTR magnitude was nonlinear and none of the 25N derivatives with Gq E MAX values <70% induced the HTR, potentially indicating that 5-HT 2A R efficacy must exceed a strong Gq-efficacy threshold level in order to induce the behavior.Similar to Gq dissociation data, the magnitude of the HTR induced by 25N derivatives is correlated with their 5-HT 2A R Gq/11mediated calcium flux response (R S = 0.8175, p = 0.0002; Supplementary Fig. 10A) and only the compounds with Gq-mediated calcium flux efficacy exceeding 70% 5-HT E MAX induced head twitches, providing further evidence that 5-HT 2A R Gq-efficacy is necessary for psychedelic potential.
We also tested whether a similar relationship exists for drug potencies (ED 50 ) in HTR experiments.Although the in vivo potencies of psychedelic drugs are known to be correlated with their in vitro potencies at 5-HT 2A R [43][44][45] , it is not clear whether a similar relationship exists for the HTR.Establishing a potency relationship would further support HTR construct validity for predicting human psychedelic action via 5-HT 2A R. Notably, we observed robust and highly significant correlations between potencies in the HTR assay and in vitro 5-HT 2A R potency measures, including binding affinities (K i ) measured using [ 3 H]-ketanserin, and functional potencies (EC 50 ) for activating Gq and β-arrestin2 via 5-HT 2A R (Supplementary Fig. 10B-F).Similar correlation results were obtained for 5-HT 2A R Gq and β-arrestin2 pathways, which is not surprising because the rankorder potencies of 5-HT 2A R agonists for Gq and β-arrestin2 are similar, whereas agonist efficacies often diverge across those two pathways.Overall, these results are consistent with the known role of 5-HT 2A R in the HTR and show a good correlation between in vitro and in vivo potency measures, but they do not link the HTR to a particular transducer pathway because they fail to account for 5-HT 2A R efficacy at each respective transducer.
Encouraged by results obtained with the 25N series, we examined whether the magnitude of the HTR produced by other psychedelics is correlated with their 5-HT 2A R Gq-efficacy.To test this hypothesis, we compared 5-HT 2A R efficacies and HTR magnitude for 24 phenethylamine psychedelics (Supplementary Fig. 11; Supplementary Tables 11, 12  and 17, 18), which tend to have greater 5-HT 2 selectivity compared to other scaffolds 10 .Our results show a significant positive relationship between HTR magnitude and 5-HT 2A R Gq-efficacy (R S = 0.7339, p < 0.0001; Fig. 5C).Conversely, there is not a significant correlation between HTR magnitude and 5-HT 2A R β-arrestin2-efficacy (R S = 0.34, p = 0.104; Fig. 5D).Consistent with the 25N series data, all of the tested psychedelics activated Gq with relatively high efficacy and the trendline between HTR magnitude and Gq-efficacy indicates a minimum of 70% 5-HT 2A R Gq-efficacy (%5-HT E MAX ).In summary, our results indicate there is a threshold level of 5-HT 2A R Gq-efficacy required for psychedelic-like activity.

Phenethylamines Phenethylamines
To directly test the involvement of 5-HT 2A R-Gq signaling in the HTR, we evaluated whether inhibition of Gq signaling can block the HTR.Because the use of signaling inhibitors can potentially be confounded by off-target effects, we targeted two different proteins within the Gq-PLC effector pathway to generate convergent evidence.YM-254890 selectively inhibits Gq/11 46 and is effective in mice when administered systemically or centrally 47 .Intracranial (ICV) pretreatment with YM-254890 blocked the HTR induced by DOI (Fig. 6H).We also tested the phosphoinositide-selective PLC inhibitor edelfosine 48 , which is brain penetrant in mice, with a brain/plasma ratio of ~0.5 after systemic administration 49 .Edelfosine blocked the HTR induced by 25N-NBOMe (4) and DOI (Fig. 6I).These results strongly support the conclusion that the HTR induced by psychedelics is dependent on activation of the 5-HT 2A R-Gq-PLC pathway.This dependence on 5-HT 2A -Gq signaling is supported by the lack of a HTR with 25N-N1-Nap (16) and other β-arrestin2-biased 5-HT 2A R agonists, and it does not appear that 5-HT 2A R β-arrestin2 recruitment is sufficient to induce head twitches.Although YM-254890 inhibited the response to DOI to a greater degree than Gq gene deletion 42 , the ability of both manipulations to dampen the response to DOI strongly supports our conclusions that 5-HT 2A R-Gq signaling is necessary for the HTR and therefore psychedelic potential.
5-HT 2A R agonists are known to cause receptor downregulation and tolerance or tachyphylaxis 65 .We tested whether β-arrestin2-biased 5-HT 2A R agonists induce tolerance in vivo, based on the rationale that tolerance could potentially act as an in vivo readout of β-arrestin2 recruitment.Our hypothesis was that repeated treatment with 25N-N1-Nap (16) would induce tolerance similar to the balanced 5-HT 2A R agonist DOI.Results were normalized to the vehicle control group to facilitate comparison of the amount of tachyphylaxis induced by the three 5-HT 2A R ligands.Once daily administration of 25N-N1-Nap (16) or DOI to mice for five consecutive days reduced the ability of a challenge dose of DOI administered 24 h later to induce the HTR (Fig. 8C, D).Conversely, the response to DOI was not altered in mice treated repeatedly with a dose of the 5-HT 2A R antagonist PIM that is capable of blocking the acute behavioral response to DOI (Fig. 8E).Hence, repeated administration of 25N-N1-Nap (16), DOI, and PIM produced effects in mice that closely parallel the in vitro internalization data.Based on these results, β-arrestin2 may play a role in the tachyphylaxis that occurs after repeated administration of 5-HT 2A R agonists.
Finally, to assess whether β-arrestin-biased 5-HT 2A R agonists may have similar therapeutic potential as 5-HT 2A R antagonists/inverse agonists, we tested whether 25N-N1-Nap (16) can antagonize PCPinduced hyperactivity in mice.5-HT 2A R antagonists are highly effective at blocking the hyperlocomotion induced by NMDA receptor antagonists such as PCP and MK-801 17,66 .25N-N1-Nap (16) blocked the hyperactivity induced by PCP in mice when tested at a dose that had no effect on baseline activity (Fig. 8F).We confirmed that the 5-HT 2Aselective antagonist M100907 has a similar effect on reducing PCPinduced hyperactivity in mice (Fig. 8G) consistent with an antipsychotic-like profile.

Discussion
Our ultimate aim was to elucidate which 5-HT 2A R transducer is responsible for psychedelic activity.Here we show that rational and structure-based design can be leveraged to develop 5-HT 2A R-selective compounds with a wide-range of functional activities, including β-arrestin2-biased agonists.Docking, MD, and mutagenesis revealed that W336 6.48 plays a key role in the mechanism of β-arrestin2-biased agonists.Finally, β-arrestin2-biased 5-HT 2A R agonists were used to probe the involvement of this transducer toward the psychedelic potential of 5-HT 2A R agonists.The ability of 5-HT 2A R agonists to induce the HTR was found to be correlated with Gq-efficacy but not β-arrestin2 recruitment.5-HT 2A R β-arrestin2-biased agonists did not induce psychedelic-like behavioral effects, but blocked psychedelic-like behaviors in vivo.Overall, we identified multiple structural and chemical features of psychedelics that can be targeted to fine-tune 5-HT 2A R activity, potentially modulating therapeutic efficacy and tolerability of 5-HT 2A R ligands for various therapeutic indications including psychosis.
Although the specific 5-HT 2A transducers mediating the HTR have not been conclusively identified, Gq/11 and β-arrestin2 are clear candidates.Previous studies in constitutive knockout (KO) mice have attempted to address the role of Gq and β-arrestin2 in the HTR but did not yield conclusive results.Gq deletion attenuated but did not eliminate the HTR induced by DOI 42 , potentially because other Gq protein subtypes such as G11 may be involved.HTR studies in β-arrestin2 KO mice have variously reported that the response to DOI is unaltered 41,67 , response to 5-MeO-DMT is enhanced 68 , or response to LSD is attenuated 40 .Although the reason for these discrepancies is unknown, it could reflect altered membrane receptor trafficking in β-arrestin2 KO mice, leading to constitutive receptor desensitization and other adaptations.Given the lack of an effect of 25N-N1-Nap (16) and other β-arrestin2-biased 5-HT 2A R agonists in the HTR paradigm, 5-HT 2A R β-arrestin2 recruitment does not appear to be sufficient to induce head twitches.Although β-arrestin2 may not be involved in the HTR, our findings do implicate β-arrestin2 in the tachyphylaxis that occurs after repeated administration of 5-HT 2A R agonists.Mice treated repeatedly with psychedelic drugs develop tolerance and cross-tolerance, potentially reflecting 5-HT 2A R downregulation 67 .Similarly, repeated treatment with 25N-N1-Nap ( 16) induced a tolerance to a subsequent challenge dose of DOI, indicating β-arrestin2-biased agonists may also be capable of inducing 5-HT 2A R downregulation.Although it was recently reported that β-arrestin2 KO mice do show tolerance to DOIinduced HTR after repeated treatment 67 , the use of constitutive KOs to address the mechanisms of GPCR tolerance is not ideal because of potential aforementioned adaptations (e.g.β-arrestin1 to offset loss of β-arrestin2).
One important result of these studies is the discovery of an apparent 5-HT 2A R Gq-efficacy threshold for induction of the HTR in mice.In addition to finding a robust and highly significant correlation between the magnitude of the HTR and multiple readouts of 5-HT 2A R Gq-efficacy, we found that compounds with a Gq E MAX < 70% do not induce the HTR.5-HT 2A R Gq-efficacy was found to be correlated with HTR magnitude in previous studies of mixed 5-HT 2A/2C agonists 69,70 , but those studies did not test enough compounds to identify a clear Gq-efficacy threshold for HTR activity.Since mice exhibit a baseline level of spontaneous HTR that is driven by basal 5-HT 2A R activation 71 , the existence of a threshold for HTR activity is not surprising because compounds with low Gq-efficacy may act as partial antagonists relative to endogenous 5-HT basal stimulation.A similar threshold may exist for psychedelic effects in humans because all psychedelics we tested activated 5-HT 2A R-Gq dissociation with E MAX > 70%, whereas non-psychedelic analogs have lower efficacy (E MAX < 70%), which likely explains why the non-psychedelics do not induce the HTR in mice or psychedelic effects in humans.Consistent with our findings, lisuride (E MAX = 48.6%)was reported to have substantially lower Gq-efficacy than LSD (Emax = 84.6%)or DOI (E MAX = 81.3%) in a 5-HT 2A R-Gq calcium mobilization assay 72 .Although we did not test the putative nonpsychedelic 5-HT 2A R agonist tabernanthalog, it reportedly has E MAX = 57% in a 5-HT 2A R calcium flux assay 5 , which may be too low to induce the HTR.These data have implications for drug development as it should be possible to identify 5-HT 2A R-Gq partial agonists that do not induce the HTR and lack strong psychedelic effects in humans but retain sufficient Gq-efficacy to induce therapeutic neurophysiological effects via 5-HT 2A R (e.g., induction of neuroplasticity).2-Br-LSD is consistent with this hypothesis because it induces neuroplasticity via 5-HT 2A R even though its 5-HT 2A R-Gq E MAX is subthreshold to induce the HTR 11 .Based on these results, it should be possible to rationally design non-psychedelic 5-HT 2A R agonists with therapeutic potential by fine-tuning 5-HT 2A R Gq-efficacy.In effect, partial 5-HT 2A R-Gq agonists may act as mixed agonist-antagonists, similar to buprenorphine and other opioid receptor partial agonists 73 , which contribute to their superior safety profile and greater tolerability 17 .Future studies will also need to examine partial 5-HT 2A R-Gq agonists in preclinical disease models to assess whether they possess therapeutic-like activity, for example, as rapid-acting antidepressants.
Based on our results, ligand interactions with residue W336 6.48 are an important predictor of agonists with weaker Gq-efficacy but do not fully explain the β-arrestin bias exhibited by these compounds.Residue W6.48 is known to be critical in propagating conformational changes by altering the position of TM6 to impact the cytosolic transducer binding pockets and influence bias 34 , but the change in W336 6.48 in our 25N-N1-Nap (16) simulation can likely influence other trigger motifs (PIF and NPxxY), which may be important to preserve GPCR-arrestin recruitment.For example, an influence of W6.48 on the PIF/PIW motif and corresponding changes in TM6 orientation were observed in MD studies with bias at β 2 -adreneregic 32 , rhodopsin 74 , MOR 34 , 5-HT 2C R 75 , S1PR1 76 , and other GPCRs 35 .Moreover, our MD results with the "partially active" TM6 orientation are intriguing given several β-arrestin1 GPCR structures, including LSD-bound 5-HT 2B R structures 77 , show a larger TM6 outward movement relative to the G protein-bound state, though differences may exist in the initial conformation recognized by the transducer and the resulting complex.Therefore, further structural studies are needed to identify other conformational changes involved in Gq versus arrestin-modulation of the binding pocket, especially with respect to these discovered biased ligands.
warranted.Second, the use of non-human model organisms raises questions about human translatability.We used mouse HTR as a predictor of human psychedelic-like activity because it has excellent predictive and construct validity for psychedelics and can distinguish non-psychedelic 5-HT 2A agonists 9,11,78 .However, species nuances may limit extrapolation of these findings to humans and the degree Gqefficacy thresholds align in humans and rodents is unclear.Finally, we chose not to use the forced swim test or tail suspension to assess antidepressant-like effects with 5-HT 2A R β-arrestin-biased agonists, because animal behavioral despair models lack translational validity [79][80][81] .Utilizing an RDoC-like approach 82 to investigate potential clinical applications will best serve to illuminate the therapeutic potential of biased or partial 5-HT 2A R agonists.The potential therapeutic utility of β-arrestin-biased 5-HT 2A R agonists needs further study, but our findings suggest they mimic psychedelics in some ways (induction of cross-tolerance and 5-HT 2A R downregulation) but also produce antagonist-like effects (e.g.blockade of PCP hyperlocomotion and DOI-induced HTR).Potentially, β-arrestin-biased ligands may mimic the therapeutic effects of 5-HT 2A R antagonists with less potential to disrupt cognition, possibly improving their efficacy and tolerability, but pharmacokinetic parameters should be investigated to ensure proper dosing and receptor occupancy in future experiments.
In conclusion, we have shown that psychedelic drugs activate both 5-HT 2A R-Gq dissociation and β-arrestin2 recruitment, which led to us to design 5-HT 2A R-selective biased agonists to probe 5-HT 2A R transducers necessary for psychedelic potential.These results indicate that a 5-HT 2A R Gq-efficacy is necessary to produce psychedelic-like effects, as measured by the HTR, and that it is possible to predict psychedelic potential based on this threshold.This study has implications for understanding the neurobiological basis of psychedelic effects and reveals strategies for designing non-psychedelic 5-HT 2A R agonists that can potentially be used as therapeutics.

Ethical statement
The mouse studies were conducted in accordance with National Institutes Health (NIH) guidelines and were approved by the University of California San Diego Institutional Animal Care and Use Committee (Protocol #S17044).

Synthesis materials and methods
N-benzyl-compounds were synthesized by reductive amination (using NaBH 4 ) of the pre-formed imine (in solution) obtained by treating the primary amine (e.g., 25N) with the respective aldehyde in dry (3 Å molecular sieves) methanol and THF in the presence of 3 Å molecular sieves in the dark under an argon atmosphere for at least 48 hours.Descriptions, including yields and analytical data including 1 H and 13 C NMR chemical shift assignments, 1 H and 13 C NMR spectra, HPLC traces are provided in the Supplementary Information.
Intermediates and reagents for synthesis were purchased from Sigma-Aldrich (St Louis, MO, USA), AKSci, and Alfa Aesar.Reagents were generally 95% pure or greater.200-proof ethyl alcohol was obtained from Pharmaco (Greenfield Global, CT, USA).Silica gel flash column chromatography was performed using Merck silica gel grade 9385 (230-400 mesh, 60 Å).Melting points were measured using a Digimelt A160 SRS digital melting point apparatus (Stanford Research Systems, Sunnyvale, CA, USA) using a ramp rate of 2 °C/min.

High-performance liquid chromatography (HPLC)
HPLC analyses were performed on an Agilent 1260 Infinity system that includes a 1260 quaternary pump VL, a 1260 ALS autosampler, a 1260 Thermostatted Column Compartment, and a DAD Multiple Wavelength Detector (Agilent Technologies, Santa Clara, CA, USA).Detection wavelengths were set at 220, 230, 254, and 280 nm but only 220 nm was used for analysis.A Zorbax Eclipse XDB-C18 analytical column (5 µm, 4.6 × 150 mm) from Agilent Technologies was used.Mobile phase A consisted of 10 mM aqueous ammonium formate buffer titrated to pH 4.5.Mobile phase B consisted of acetonitrile.The injection volume of samples was 10 µL, flow rate was 1.0 mL/min, and the column temperature was set at 25 o C. Samples were prepared by preparing a 1 mg/mL solution in 1:1 A:B.All samples were injected in duplicate with a wash in between each run.Run time was 10 minutes with a mobile phase ratio (isocratic) of 1:1 for A:B.Chromatograms were analyzed using the Agilent ChemStation Software (Agilent Technologies).Purity values were calculated from area under the curves of the absorbance at 220 nm of any resulting peaks.

High resolution mass spectrometry (HRMS)
HRMS data were obtained on a Thermo Orbitrap Exactive Mass Spectrometer with an Orbitrap mass analyzer.The instrument was calibrated using electrospray ionization with Pierce TM LTQ ESI Positive Ion Calibration Solution from ThermoFisher Scientific.Samples were introduced into the instrument and ionized via an Atmospheric Solids Analysis Probe (ASAP).Data were analyzed in the Thermo Xcalibur Qual Browser software and identity was confirmed if <5 ppm error.Measurement parameters were as follows: Aux gas flow rate-8, Spray Voltage-3.50kV, Capillary temperature-275 o C, Capillary Voltage-25.00 V, Tube Lens Voltage-65.00 V, Skimmer Voltage-14.00 V, Heater Temperature-100 o C.

Elemental analysis
Elemental analysis (C, H, N) was run on select compounds by Galbraith Laboratories, Inc. (Knoxville, TN).

Nuclear magnetic resonance
1 H (400 MHz) and 13 C NMR spectra (101 MHz) were obtained on the hydrochloride salts as a solution in anhydrous d 6 -DMSO ( ~20 mg/mL) ( > 99.9% D, Sigma-Aldrich) on a Bruker Avance III with PA BBO 400S1 BBF-H-D-05 Z plus probe (Bruker Corporation, Billerica, MA, USA).Solvent (δ = 2.50 and 39.52 ppm for 1 H and 13 C spectras respectively) was used for internal chemical shift references. 19F (376.5 MHz) NMR was run as described above using ~100 µL of trichlorofluoromethane (99%+, Sigma-Aldrich) as internal reference (δ = 0.0 ppm).Full NMR chemical shift assignments were made using chemical shift position, splitting patterns, 13 C and 13 C PENDANT or APT and hetero-and homo-2-D experimentss including HMQC or HSQC, HMBC and COSY (45°pulse tilt).A background water concentration of solvent (determined as integration ratio relative to solvent shift) was also determined to check for water content (indicative of a hydrate) within the final salts for 25N compounds.Evidence of hydrates was not observed.

Receptor binding experiments
Competition-based receptor binding studies were performed by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP) using described methods 83 .Target compounds were dissolved in DMSO and an initial screen performed to assess displacement of the radioligand at target receptors using a concentration of 10 µM.Those compounds that showed >50% displacement of specific radioligand binding for a given receptor then underwent secondary screenings at varying concentrations to determine pK i values.For the K i experiments, compounds were run in triplicate on separate plates.Each plate contained a known ligand of the receptor as a positive control.Full experimental details are available in the NIMH PDSP assay protocol book.For all 5-HT subtypes, an N = 3-4 was performed except where noted (Supplementary Table 8) and a mean pK i and SEM was calculated using these replicate experiments.Affinities at off-targets are provided in Supplementary Tables 9, 10.

Gq-dissociation and β-arrestin2 recruitment BRET assays
To measure 5-HT receptor-mediated β-Arrestin2 recruitment as measured by BRET 1 , HEK293T cells (ATCC Cat# CRL-11268; mycoplasma-free) were subcultured in DMEM supplemented with 10% dialyzed FBS (Omega Scientific) and were co-transfected in a 1:15 ratio with human or mouse 5-HT receptors containing C-terminal fused renilla luciferase (RLuc8), and a Venus-tagged N-terminal β-arrestin2 using 3:1 ratio of TransiT-2020 (Mirus).5-HT 2A receptor mutants were designed and performed using Q5 mutagenesis kit (New England Biolabs).All DNA was sequence verified using Sanger nucleotide sequencing (Retrogen, San Diego, CA).To measure 5-HT receptor-mediated Gq activation via Gq/γ1 dissociation as measured by BRET 2 , HEK293T cells were subcultured in DMEM supplemented with 10% dialyzed FBS and were co-transfected in a 1:1:1:1 ratio with RLuc8-fused human Gαq (Gαq-RLuc8), a GFP 2 -fused to the C-terminus of human Gγ1(Gγ1-GFP 2 ), human Gβ1, and 5-HT receptor using TransiT-2020 84 .After at least 18-24 hours, transfected cells were plated in poly-lysine coated 96-well white clear bottom cell culture plates in DMEM containing 1% dialyzed FBS at a density of 25-40,000 cells in 200 µl per well and incubated overnight.The next day, media was decanted and cells were washed with 60 µL of drug buffer (1× HBSS, 20 mM HEPES, pH 7.4), then 60 µL of drug buffer was added per well.Cells were pre-incubated at in a humidified atmosphere at 37 °C before receiving drug stimulation.Drug stimulation utilized 30 µL addition of drug (3X) diluted in McCorvy buffer (1× HBSS, 20 mM HEPES, pH 7.4, supplemented with 0.3% BSA fatty acid free, 0.03% ascorbic acid) and plates were incubated at indicated time and temperatures.Substrate addition occurred 15 minutes before reading and utilized 10 µL of the RLuc substrate, either coelenterazine h for β-Arrestin2 recruitment BRET 1 or coelenterazine 400a for Gq dissociation BRET 2 (Prolume/Nanolight, 5 µM final concentration) and was added per well.Plates were read for luminescence at 485 nm and fluorescent eYFP emission at 530 nm for BRET 1 and at 400 nm and fluorescent GFP 2 emission at 510 nm for BRET 2 at 1 second per well using a Mithras LB940 (Berthold) or a PheraStar FSX (BMGLabTech).The BRET ratios of fluorescence/luminescence were calculated per well and were plotted as a function of drug concentration using Graphpad Prism 5 or 9 (Graphpad Software Inc., San Diego, CA).Data were normalized to % 5-HT stimulation and analyzed using nonlinear regression "log(agonist) vs. response" to yield Emax and EC 50 parameter estimates.

Calcium flux assays
Stably expressing 5-HT receptor Flp-In 293 T-Rex Tetracycline inducible system (Invitrogen Cat#R78007, mycoplasma-free) were used for calcium flux assays 85 .Cell lines were maintained in DMEM containing 10% FBS, 10 µg/mL Blasticidin (Invivogen), and 100 µg/mL Hygromycin B (GoldBio).Day before the assay, receptor expression was induced with tetracycline (2 µL/mL) and seeded into 384-well poly-L-lysinecoated black plates at a density of 7,500 cells/well in DMEM containing 1% dialyzed FBS.On the day of the assay, the cells were incubated with Fluo-4 Direct dye (Invitrogen, 20 µl/well) for 1 h at 37 °C, which was reconstituted in drug buffer (20 mM HEPES-buffered HBSS, pH 7.4) containing 2.5 mM probenecid.After dye load, cells were allowed to equilibrate to room temperature for 15 minutes, and then placed in a FLIPR TETRA fluorescence imaging plate reader (Molecular Devices).Drug dilutions were prepared at 5X final concentration in drug buffer (20 mM HEPES-buffered HBSS, pH 7.4) supplemented with 0.3% BSA fatty-acid free and 0.03% ascorbic acid.Drug dilutions were aliquoted into 384-well plastic plates and placed in the FLIPR TETRA (Molecular Devices) for drug stimulation.Fluorescence for the FLIPR TETRA were programmed to read baseline fluorescence for 10 s (1 read/s), and afterward 5 µl of drug per well was added and read for a total of 5-10 min (1 read/s).Fluorescence in each well was normalized to the average of the first 10 reads for baseline fluorescence, and then either maximum-fold peak increase over basal or area under the curve (AUC) was calculated.Either peak or AUC was plotted as a function of drug concentration, and data were normalized to percent 5-HT stimulation.Data was plotted and non-linear regression was performed using "log(agonist) vs. response" in Graphpad Prism 9 to yield Emax and EC 50 parameter estimates.

Surface expression/internalization experiments
Surface expression was measured using a HiBit-tagged 5-HT 2A receptor and the Nano-Glo HiBit Extracellular Detection System (Promega).N-terminal HiBit-tagged human 5-HT 2A receptor was cloned into pcDNA3.1 using Gibson Assembly.HEK293T cells (ATCC Cat#CRL-11268; mycoplasma-free) were transfected into 10-cm tissue culture dishes in a 1:15 ratio of HiBit-tagged human 5-HT 2A receptor: human β-Arrestin2 (cDNA Resource Center; www.cDNA.org).Cells were transfected in DMEM 10% dFBS and the next day, cells were plated into either poly-L-lysine-coated 96-well white assay plates (Grenier Bio-One).On the day of the assay, plates were decanted and HEPESbuffered DMEM without phenol-red (Invitrogen) was added per well.Plates were allowed to equilibrate at 37 °C in a humidified incubator before receiving drug stimulation.Compounds (including 5-HT as control) were serially diluted in McCorvy buffer (20 mM HEPESbuffered HBSS, pH 7.4 supplemented with 0.3% BSA fatty-acid free and 0.03% ascorbic acid), and dilutions were added to plates in duplicate (96).Plates were allowed to incubate at 37 °C for 1 hour in a humidified incubator or a specified time point.Approximately 15 minutes before reading, LgBit and coelenterazine h (5 uM final concentration) were added to each well.Plates were sealed to prevent evaporation and read on either a PheraStar FSX (BMB Lab Tech) or Mithras LB940 (Berthold Technologies) at 485 nm at 37 °C for time-capture quantification of internalization or loss of surface expression.Luminescence was plotted as a function of drug concentration using Graphpad Prism 5 or 9 (Graphpad Software Inc., San Diego, CA).Data were analyzed using nonlinear regression "log(agonist) vs. response" to yield Emax and EC 50 parameter estimates and normalized to % 5-HT surface expression, which a full concentration-response curve was present on every plate.

Induced fit docking
Docking simulations of six of the 25N ligands, selected to assess the binding modes and provide potential insight into the SAR around transducer or receptor selectivity, were carried out against the active state human 5-HT 2A receptor (PDB: 6WHA) using the Induced-Fit Docking (IFD) protocol 86 of the Schrodinger Suite (2020a).IFD was run with extended sampling enabled, using the default setting of residues within 5 Å of the experimental ligand (25N-NBCN) and other parameters maintained at their default values, and ligand structures processed using the LIGPREP tool (target pH of 7) of the Schrödinger (release 2020c).
Poses generated in the IFD runs were analyzed with PyMol visualization application (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

Estimation of ring substituent effects on molecular electrostatic potential
A series of model compounds were constructed that included only the benzylamine or naphthylamine portion of the corresponding ligand structure.Quantum mechanical optimization of the model compounds, in both neutral and cationic forms, was carried out using density functional theory (DFT) with the Jaguar tool of the Schrodinger suite using the B3LYP functional and 6-311 g**++ basis set, with the exceptions of the models for 25N-NBI, where the halogen atom was not supported, and a smaller basis (3-21 g*++) was substituted.We computed Hirshfeld partial atomic charges (Supplementary Table 4) for the model compounds using the optimized wavefunctions as a proxy for overall MEP.Hirshfeld charges are computed from a spatial partitioning of the electron density, and have been shown to correlate well with a variety of properties of aromatic molecules 87 .

Molecular dynamics (MD) simulations
Simulations were performed for each of two ligands (25CN-NBOH or 25N-N1-Nap (16) in complex with the human 5-HT 2A serotonin receptor in a lipid bilayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and SPC water with enough Cl -and Na + ions to neutralize the system at biologic salinity (0.15 M ionic strength).For the 25CN-NBOH system a model of the protein-ligand part of the system was made from chain A of the 6WHA PDB entry, downloaded from the OPM server 88 .Missing extracellular loops were filled in with those from chain A of PDB entry 6WGT.Schrödinger's Protein Preparation Wizard 89 was employed to use Prime 90 to fill in missing side chains, and to use Epik 91 to select the appropriate tautomers and protonation states of the protein and ligand.Due to anticipated difficulty modeling the long ICL3, the helices of TM5 and TM6 were capped, along with the N and C termini, with polar terminating residues: COOH for residue Q262 5.66 and NH 2 for I315 6.27 .During pilot simulations, entanglement of polar sidechains in the membrane occurred.To prevent this, it was found necessary that the rotamers for the loops and capped termini whose sidechains would otherwise interact with the polar heads of the lipid molecules be manually adjusted to ensure adequate water solvation at the beginning of the simulation.This was achieved by maximizing the magnitude of atomic coordinate z of terminal side-chain atoms, where the z-axis is perpendicular to the membrane and z = 0 locates the middle of the bilayer.Another issue identified during preliminary simulations was that DPPC tails would intrude into the orthosteric pocket between helices TM4 and TM5, causing the nitrile end of the ligand to project up toward the extracellular side of the pocket.On careful inspection, it was discovered that there is a stable position for a water molecule among residues D120 2.50 , S162 3.39 , and N376 7.49 .Manual introduction of a solvent molecule into this void volume successfully prevented lipid intrusion.
A disulfide bond between C148 3.25 and C227 45.50 18 was added to the topology, as well one between C349 6.61 and C353 ECL3 .Each ligand was protonated at the basic nitrogen (as confirmed by Epik) and topology parameter files needed for subsequent dynamics simulation were determined using the ATB server [https://atb.uq.edu.au/].Partial atomic charges for the ligands were computed via Schrödinger's Jaguar tool using the density-functional method (B3LYP-D3 functional, 6-31 G** basis), with discrete charges derived from the geometryoptimized wavefunction using the Hirshfeld approach (Supplementary Table 4).The total protein comprised 278 residues with 2849 atoms.The remaining system consisted of 6933 water molecules, 29 Cl -ions, 20 Na + ions, and 88 DPPC molecules.25CN-NBOH has 44 atoms.The total number of atoms in the system is 28,137.The orthorhombic initial system dimensions were 61.319 × 60.911 × 97.412 Å 3 .
The protein-ligand complex for 25N-N1-Nap (16) starts with the structure from the top-ranked pose determined by induced fit docking (IFD).A chimeric homology model was created in Maestro using the IFD structure as the template everywhere except at the missing loops; these are taken from the 25CN-NBOH-system.This resulting protein-25N-N1-Nap (16) homology model was superposed (using the positions of the TM C α s) onto the protein-ligand complex in the full (water-ionlipid-protein-ligand) unrelaxed construct built for the 25CN-NBOH simulation.The protein-25CN-NBOH complex was then removed and the 25N-N1-Nap (16)-protein complex substituted in its place.As the IFD docking involved minimal changes to backbone conformation, the resulting model for the 25N-N1-Nap (16) ligand had helix and loop positions nearly identical to those of the 25CN-NBOH model, permitting a direct structural substitution.That said, prior to any further computations, a restrained minimization was carried out with only the membrane/solvent system fully mobile, to remove any high-energy interatomic overlaps that might have been inadvertently produced by the alignment and substitution.The 25N-N1-Nap (16) simulation comprised a total of 28,143 atoms (50 atoms for the ligand and otherwise identical to the 25CN-NBOH system).The orthorhombic initial system dimensions were 61.864 × 61.453 × 96.12 Å 3 .

Assessment of the head-twitch response
The head-twitch response (HTR) was assessed using a head-mounted neodymium magnet and a magnetometer detection coil 78 .The mice were allowed to recover from the magnet implantation surgeries for at least 1 week prior to behavioral testing.Mice were tested in multiple HTR experiments, with at least 7 days between studies to avoid carryover effects.HTR experiments were conducted in a well-lit room, and the mice were allowed to habituate to the room for at least 1 h prior to testing.Mice were tested in a 12.5-cm diameter glass cylinder surrounded by a magnetometer coil.Coil voltage was low-pass filtered (2 kHz), amplified, and digitized (20-kHz sampling rate) using a Powerlab (model /8SP or 8/35) with LabChart software (ADInstruments, Colorado Springs, CO, USA).Head twitches were identified in the recordings off-line by their waveform characteristics 94 or using artificial intelligence 95 .HTR counts were analyzed using one-way ANOVAs or one-way Welch ANOVAs (in cases where groups showed unequal variances).Tukey's test or Dunnett's T3 multiple comparisons test was used for post hoc comparisons.Significance was demonstrated by surpassing an α level of 0.05.Median effective doses (ED 50 values) and 95% confidence intervals for dose-response experiments were calculated by nonlinear regression (Prism 9.02, GraphPad Software, San Diego, CA, USA).

Assessment of PCP-induced locomotor activity
The mouse behavioral pattern monitor (BPM) was used to assess locomotor activity 96 .Each mouse BPM chamber (San Diego Instruments, San Diego, CA, USA) is a transparent Plexiglas box with an opaque 30 × 60 cm floor, enclosed in a ventilated isolation box.The position of the mouse in x,y coordinates is recorded by a grid of 12 × 24 infrared photobeams located 1 cm above the floor.A second row of 16 photobeams (parallel to the long axis of the chamber, located 2.5 cm above the floor) is used to detect rearing behavior.Holepoking behavior is detected by 11 1.4-cm holes that are situated in the walls (3 holes in each long wall, 2 holes in each short wall) and the floor (3 holes); each hole is equipped with an infrared photobeam.The status of each photobeam is sampled every 55 ms and recorded for offline analysis.Mice were allowed to habituate to the testing room for at least 1 h prior to testing.For the BPM experiments, mice (n = 6/group) were pretreated SC with test drug or vehicle, PCP (5 mg/kg) or vehicle was injected IP 10 minutes later, and then the mice were placed in the BPM chambers 10 minutes after the second injection and activity was recorded for 60 minutes.Locomotor activity was quantified as distance traveled, which was analyzed in 20-minute blocks using a threeway ANOVA, with pretreatment and treatment as between-subject variables and time as a within-subject variable.Tukey's test was used for post hoc comparisons.Significance was demonstrated by surpassing an α level of 0.05.
Effect of Repeated Treatment with 5-HT 2A receptor ligands on Head-Twitch Response In the first experiment, mice (n = 6-7/group, 19 total) were injected SC (5 mL/kg) once daily with vehicle (water containing 5% Tween-80), 25N-N1-Nap hydrochloride (20 mg/kg), or (±)-DOI hydrochloride (10 mg/kg) for five consecutive days.Twenty-four hours later, all of the mice were challenged with an IP injection of 1 mg/kg (±)-DOI hydrochloride and then HTR activity was recorded for 40 minutes.In the second experiment, mice (n = 6/group, 12 total) were injected IP (5 mL/kg) once daily with vehicle (water) or pimavanserin hemitartrate (1 mg/kg) for five consecutive days.Twentyfour hours later, all of the mice were challenged with an IP injection of 1 mg/kg (±)-DOI hydrochloride and then HTR activity was recorded for 40 minutes.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig. 1 | Psychedelics exhibit similar Gq and β-arrestin2 activity at 5-HT 2A R. Schematic of 5-HT 2A receptor G protein dissociation (A) and β-arrestin recruitment (B) to determine transducer preferences.(C) Determination of 5-HT 2A receptor G protein-wide and β-arrestin1/2 transducer preferences as estimated by magnitude net BRET for each transducer as stimulated by 5-HT.Data represent the mean and SEM from three independent experiments, which were performed at 37 °C with 60-minute compound incubations.D-K Comparison of 5-HT 2A receptor Gq dissociation (red) and β-arrestin2 recruitment (blue) for 5-HT (D) and several classes of psychedelics: (E) Psilocin, (F) DMT, (G) 5-MeO-DMT, (H) 2C-I, (I) DOI, (J) 25I-NBOMe, K LSD.Data represent the mean and SEM from three independent experiments, which were performed at 37 °C with 60-minute compound incubations.L Heat map of Gq dissociation and β-arrestin2 recruitment kinetics displayed as log (E MAX /EC 50 ) for the psychedelics tested.Data represent the mean and SEM from three independent experiments, which were performed at 37 °C at the indicated compound incubation time points.Source data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | Psychedelic potential is correlated with 5-HT 2A -Gq signaling.A Scatter plot showing the relationship between 5-HT 2A receptor Gq-efficacy (E MAX values) and head-twitch response (HTR) magnitude (maximum counts per minute induced by the compound) for 14 members of the 25N series.Spearman's rank correlation coefficient R s is shown.The regression line generated by fitting the data using nonlinear regression is included as a visual aid.B Scatter plot showing the relationship between 5-HT 2A receptor β-arrestin2 recruitment efficacy (E MAX values) and HTR magnitude for 14 members of the 25N series.C Scatter plot showing the relationship between 5-HT 2A receptor Gq-efficacy (E MAX values) and HTR magnitude for 24 phenethylamine psychedelics.To illustrate the non-linear nature of the relationship, the regression line generated by fitting the data using non-linear regression is shown.D Scatter plot showing the relationship between 5-HT 2A receptor β-arrestin2 recruitment efficacy (E MAX values) and HTR magnitude for 24 phenethylamine psychedelics.Source data are provided as a Source Data file.